Accumulating evidence now reveals that transcription elongation is not a straightforward read-out of the downstream DNA sequence. Co-transcriptional processing events dictate the covalent nature and fate of RNA transcripts (Moore, M. J. & Proudfoot, N.J. Cell 136, 688-700, 2009). Indeed many transcripts are targeted co-transcriptionally for rapid degradation and hence are effectively invisible to approaches that monitor mature messages (Preker, P. et al. Science 322, 1851-1854, 2008; Xu, Z. et al. Nature 457, 1033-1037, 2009; Neil, H. et al. Nature 457, 1038-1042, 2009). In addition to these processing events, the strong propensity of RNAP to pause creates barriers to elongation and provides an opportunity for regulation and coordination of co-transcriptional events (Rougvie, A. E. & Lis, J. T. Cell 54, 795-804, 1988; Proshkin, S., et al. Science 328, 504-508, 2010). In vitro, RNAP pausing is found to be ubiquitous (Kassayetis, G. A. & Chamberlin, M. J. J Biol Chem 256, 2777-2786, 1981). Elegant biophysical approaches have provided a structural and energetic understanding of RNAP pausing which results from both intrinsic properties of the polymerase itself as well as interactions with its DNA template including the presence of bound proteins (e.g. histones) (Shaevitz, J. W., et al. Nature 426, 684-687, 2003; Herbert, K. M. et al. Cell 125, 1083-1094, 2006; Hodges, C., et al. Science 325, 626-628, 2009; Kireeva, M. L. & Kashlev, M. Proc. Natl. Acad. Sci. USA 106, 8900-8905, 2009; Kireeva, M. L. et al. Mol. Cell 18, 97-108, 2005). In the cell, elongation factors likely alter the energetic landscape of transcription, but the extent and mechanism of RNAP pausing in eukaryotic cells remain largely unknown. Bridging the divide between in vivo and in vitro transcriptional views requires approaches that visualize transcription with comparable precision afforded by in vitro transcriptional assays. More generally, the ability to quantitatively monitor nascent transcripts would provide broad insights into the roles and regulation of transcription initiation, elongation and termination in gene expression.
Historically, two strategies have been used to provide snapshots of transcriptional activity in vivo. In the first approach, RNAP is crosslinked to DNA and RNAP-bound DNA elements are identified by microarrays or deep sequencing (Kim, T. H. et al. Nature 436, 876-880 (2005); Lefrançois, P. et al. BMC genomics 10, 37 (2009)). While providing a global view of RNAP binding sites, these measurements are of limited spatial and temporal resolution and do not reveal the identity of the transcribed strand or even if RNAP molecules are engaged in transcription. In the second approach, transcription is halted in vivo and then reinitiated in isolated nuclei under conditions that allow labeling of nascent chains thereby enabling them to be distinguished from bulk RNA (Core, L. J., et al. Science 322, 1845-1848 (2008); Rodriguez-Gil, A. et al. The distribution of active RNA polymerase II along the transcribed region is gene-specific and controlled by elongation factors. Nucleic Acids Research (2010)). Such “nuclear run-on” strategies reveal actively transcribed DNA regions but require extensive manipulations that limit resolution and depend on the efficient reinitiation of transcription under non-physiological conditions.